Reprogramming cellular metabolism is a hallmark of malignancy. Understanding the metabolic transitions that accompany transformation will produce new insights into the biological basis of transformation, and may generate new targets for therapy and diagnostic imaging in cancer. We use a combination of nuclear magnetic resonance spectroscopy, mass spectrometry and nanotechnology to analyze tumor cell metabolism in culture, in mouse models of cancer, and in human patients. Analysis of a large number of cultured cell lines uncovered a surprising amount of cell-autonomous metabolic heterogeneity, but also demonstrated that most cancer cells can be stratified into a finite subset of metabolic platforms defined by utilization of glucose and glutamine, two abundant extracellular nutrients that account for the bulk of carbon and nitrogen metabolism in cancer cells. We have now developed isotope tracing methods to probe intermediary metabolism in culture models that more faithfully recapitulate cell growth in vivo, and to probe metabolism directly in live tumors in mice and humans. This work revealed utilization of many of the same core activities as those observed in conventional culture, including glucose oxidation, anaplerosis, and amino acid synthesis. There were also striking differences that presumably reflect a switch to alternative pathways induced by microenvironmental factors. Ongoing work seeks to develop methods to translate these findings into novel imaging approaches to diagnose cancer and monitor the response to therapy.